Prostate cancer diagnosis and treatment

ABSTRACT

The present invention relates to novel antibodies and their use for detecting, imaging, staging, treating and monitoring of prostate cancer, and/or metastasis thereof. The present invention also relates to novel pharmaceutical compositions for the treatment of prostate cancer. Furthermore the present invention relates to assay systems and kits for detecting, imaging, staging, treating and monitoring of prostate cancer, and/or metastasis thereof.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to novel antibodies and their use for detecting,imaging, staging, treating and monitoring of prostate cancer, and/ormetastasis thereof. Furthermore, the invention also relates to novelpharmaceutical compositions for the treatment of prostate cancer.

(b) Description of Prior Art

The prostate gland is affected by various significant pathologicalconditions as benign growth (BPH), infection (prostatitis), andneoplasia (prostate cancer).

Prostate cancer is the second most frequently diagnosed cancer inCanadian and American men, after non-melanoma skin cancer, which israrely fatal. More importantly, after lung cancer, prostate cancer isthe most common cause of cancer-related death. The risk of developingprostate cancer increases significantly with age, particularly for menover 50. For men under 50 years of age the disease is uncommon and deathfrom it is rare.

Prostate cancer accounts for an estimated 28% of newly diagnosed cancerin Canadian men and more than 12% of cancer-related deaths. The currentlifetime risk of a Canadian man being diagnosed with prostate cancer isabout 1 in 8. In the United States, prostate cancer accounts forapproximately 32% of male cancer diagnoses and 14% of cancer deaths.Studies in the United States suggest that the incidence rate may beapproaching 1 in 6 men.

Because the incidence of prostate cancer increases with age, it is clearthat the burden of this illness will increase dramatically in the comingdecades. The aging of the population, particularly the baby boomers,will have important long-term implications for the number of new casesdiagnosed. Demographic trends in the next two decades will increase thepopulation at risk for prostate cancer. Statistics Canada projectionsindicate that the population of men over age 50 will increase from 3.9million in 1999 to 5.6 million in 2011 (44% increase) and 6.3 million in2016 (62% increase). The United States Census Bureau projectionsindicate that the population of men over age 50 will increase, from 33.8million in 1999 to 45.8 million in 2011 (36% increase) and 50.7 millionin 2016 (50% increase). The American Cancer Society predicts that therewill be about 180,400 new cases of prostate cancer in the United Statesin the year 2000, and about 31,900 American men will die of the disease.

As a consequence of the expected increases in the number of cases ofprostate cancer in the coming years due to rising incidence rates andthe aging North American population, more resources will likely beallocated to screening men over 50 for this condition, thereforeyielding an increase in the number of cases of identified prostatecancer.

Prostate cancer often exhibits a long latency period. However, it isbelieved that prostate cancer often remains undetected. Also, because itpossesses a high metastatic potential to bone and the lymph nodes, with<10% of individuals diagnosed with prostate cancer also demonstrated, byradionuclide scans, to have bone metastasis, prompt detection andtreatment is needed to limit mortality caused by this disease. A recentreview of treatment of prostate cancer is by Pirtskhalaishvilig et al.(2001, Cancer Practice 9(6):295).

Increased detection of prostate cancer is due in part to increasedawareness and the widespread use of clinical markers such as prostatespecific antigen (PSA). Prostate specific antigen is a protein that isproduced in very high concentrations in prostate cancer cells. Cancerdevelopment results in an altered and subsequent loss of normal glandarchitecture. This in turn leads to an inability to remove secretionsand thus the secretions reach the serum. Serum PSA measurement is onemethod for screening for prostate cancer.

The current diagnostic and treatment paradigm for prostate cancer isreflected in Clinical Practice Guidelines that are widely available topracticing physicians. The guidelines presented below outline the commonapproach to the detection and management of prostate cancer.

-   -   The Prostate Specific Antigen test is a blood test used to        detect prostate cancer in the earliest stages and should be        offered annually to men 50 and older with a life expectancy of        10 years or more, and to younger men at high risk for prostate        cancer.    -   The Digital Rectal Exam (DRE) is a test that helps to identify        cancer of the prostate, and should be performed on men who are        50 and older and to younger men at high risk for prostate        cancer.    -   A biopsy is recommended for all men who have an abnormal PSA or        DRE.    -   The options for primary management of prostate cancer are        surgery, radiation therapy or close observation. Treatment        decisions are based on the aggressiveness of the cancer, the        stage of the cancer and the life expectancy of the individual.    -   Advanced prostate cancer is best managed with hormone therapy.    -   Radiation therapy can include external and implanted seeds, a        procedure known as brachytherapy.

The PSA test, which facilitates early detection of prostate cancer, hasbeen available in Canada since 1986, although its use did not becomewidespread until the early 1990's. In 1994 the U.S. Food and DrugAdministration (FDA) approved the use of the PSA test in conjunctionwith DRE as an aid in detecting prostate cancer. The free PSA test(fPSA), a more sensitive test for prostate cancer risk than the standardPSA test, received FDA approval in 1998.

Prostate Specific Antigen is an enzyme made by all prostate cells andnormally secreted into semen. Both cancer and a number of benignconditions can change the architecture of the prostate gland so theenzyme escapes into the bloodstream. Once there, PSA can exist in twoforms, one that is free-floating and another that is bound to proteins.The standard PSA test measures both forms. There are a number ofspecialized PSA tests which are used to help differentiate betweenelevated PSA due to benign conditions and those elevations due toprostate cancer. The free PSA test evaluates the ratio between the PSAthat is free in the blood and the total PSA (free and protein bound PSA)in the blood. When the result of the free PSA test is low (i.e. <15%),there is a higher potential that the individual has prostate cancer. ThePSA velocity is used to describe the speed at which the PSA valueincreases over a series of blood tests. The PSA density is used toevaluate the level of PSA in relation to overall size of the prostategland.

The various PSA tests share some common limitations:

-   -   The principal concern is that although diagnostic accuracy has        improved with each of the modifications to total serum PSA        measurement, none of the forms is specific for prostate cancer.    -   Each requires a trade-off in specificity for increased        sensitivity and vice versa. This trade-off appears to be most        advantageous with the proportion of free PSA.    -   Elevation of PSA may indicate prostate cancer. However, several        other common benign conditions, including Benign Prostatic        Hyperplasia (BPH), are known to be associated with an elevated        PSA.

Because of the limitations of the PSA test (lack of specificity forprostate cancer and a significant number of “false positive” and “falsenegative” test results) it remains an investigational tool as opposed toan absolute diagnostic test. Abnormal findings following theadministration of the PSA test lead the investigator to perform abiopsy. Physicians are advised to consider a biopsy to confirm aprostate cancer diagnosis when a PSA test reading is at least 4.0 ng/mL,when the PSA level of an individual significantly increases from onetest to the next, or when a DRE is abnormal. A biopsy is recommended forall men who have a PSA test result above 10 ng/mL.

The limitations of the PSA test are obvious considering the fact thatonly one of four individuals biopsied receives results that are positivefor the presence of cancerous cells. A Canadian study has estimated thepositive predictive value of the PSA test to be as low as 14.4%. This issignificant considering the costs associated with a follow-up biopsy aswell as the unnecessary pain and anxiety caused for individuals.

Since FDA approval in the U.S., the fPSA test is becoming a follow-uptest for men whose PSA falls in a “diagnostic gray zone” of moderatelyelevated levels (4 to 10 ng/mL).

The digital rectal examination is a simple, inexpensive and directmethod of assessing the prostate, but it is unreliable as a soleindicator of prostate cancer. The cancer detection rate is higher withPSA screening than with digital rectal examination (DRE), and the rateincreases when the DRE modality is combined with PSA analysis and/ortransrectal ultrasound examination (TRUS). DRE has never been shown tobe reliable for, staging of prostate cancer. TRUS guided biopsy isrequired to follow-up on a positive PSA test in order to help confirmthe presence or absence of disease in the individual's prostate.

Prostate biopsies are performed to confirm the presence of cancer cellsfollowing suspicion raised by the DRE or a positive PSA test. The mostcommonly reported complications of biopsy consist of traces of blood inthe urine, semen or feces. These complications are limited and subsidewith 2-3 weeks after the procedure. Pain at the time of biopsy isuniversally reported. Only in exceptional cases is analgesia or sedationrequired. Most men (>90%) have no significant pain after 24 hours of thebiopsy. Prostate biopsies are costly in the U.S. and may be painful orpsychologically traumatic. Prostatic biopsy represents the cornerstoneof prostate cancer diagnosis.

For prostate cancers in general, biopsies miss cancers at a rateestimated as high as 50 percent. Furthermore, even if a cancer isdetected, the location and staging of cancerous cells are not adequatelyidentified.

Thus, there is a need for an improved method for diagnosis and/ordetection of cancerous prostate cells.

An important prognostic factor is prostate cancer stage. Cancer stagingis performed to determine the extent and spread of cancer in theprostate. Prostate cancer metastasizes by local spread to the pelviclymph nodes, seminal vesicles, urinary bladder, or pelvic side walls andto distant sites such as bone, lung, liver, or adrenals. Thetumor-nodes-metastasis (TMN) staging system is the one most widely usedin North America.

The limitations of the biopsy in detecting disease and staging amalignancy is compounded by the fact that prostate cancer is aheterogeneous disease with apparently independent foci of cancer scatterthroughout the gland. The cancer foci have different malignantpotentials and do not pose equal risks for the individual. Heterogeneityconfounds the interpretation of positive prostate biopsies since it isnot possible to be certain that the most clinically relevant foci ofcancer have been detected.

Approximately only 30% of early stage disease will progress toclinically relevant disease within the lifetime of the individual. It istherefore critical to be able to identify those individuals at risk ofprogression who would benefit from aggressive therapy while sparinglow-risk individuals the morbidity resulting from aggressive treatmentof indolent disease. Neither rising PSA nor the presence of cancer cellson biopsy may indicate definitively the presence of lethal disease.

Serum PSA is a valuable cancer marker but cannot be used alone todetermine the clinical or pathological stage of prostate cancer or toidentify individuals with potentially curable disease. The combinationof serum PSA with Gleason Score (a grading system for the classificationof adrenocarcinoma of the prostate by observation of the pattern ofglandular differentiation) and clinical stage provides a betterprediction of the final pathologic stage than do any of these variablesseparately. Nomograms have been developed and revised to predict thefinal pathologic stage based on a combination of serum PSA level,Gleason Score, and clinical stage. Because these nomograms only offer astatistical probability of disease organ confinement, furtherradiographic evaluation has often been used for the individual. However,definitive detection of lymph node metastases with standard anatomicalmodalities of computed tomography (CT) and magnetic resonance imaging(MRI) has generally proved ineffective, except for the increasingly moreuncommon cases with large volume soft-tissue involvement (greater than 1cm) at presentation.

There is a great need for a new prostate imaging technology thatprovides for accurate visualization of extraprostatic growth indicativeof metastasis. Such a technology would provide physicians with a tool todetermine the progression of the cancer and would be extremely valuablein directing treatment options. Spectroscopy significantly improves thediagnosis of extracapsular extension by MRI. However, studiesdemonstrate that there is high variability in how clinicians interpretthe significance of extracapsular extension. Both CT and MRI can behelpful in staging prostate cancer, because they can indicateperiprostatic cancer spread, lymph node abnormality and boneinvolvement, but their sensitivity for revealing cancer extension haslimitations.

Imaging techniques such as CT or MRI are unable to distinguishmetastatic prostate cancer involvement of lymph nodes by criteria otherthan size (i.e. >1 cm). Thus, these imaging techniques, being inherentlyinsensitive and non-specific, are insufficient for detection of disease.

The presence of pelvic lymph node metastasis influences both thetreatment and the prognosis of individuals with prostate cancer. Lymphnode involvement can be assessed surgically. However, incompletesampling at the time of radical prostatectomy lead's to false-negativeinterpretations in at least 12%, and possibly as many as, 33% ofindividuals with lymph node metastases, because isolated metastases inthe external iliac, presciatic, or presacral lymph nodes are outside theboundaries of the standard Pelvic Lymph Node Dissection.

Thus, there is a need for a non-invasive test that is able to identifylymph node metastases in individuals at risk for extraprostatic diseasefollowing the detection of elevated PSA and/or abnormal DRE and apositive biopsy. This will allow clinicians to reliably differentiateindividuals with organ-confined disease from those with metastaticspread to lymph nodes. This will provide the opportunity for theindividual and physician to make an informed decision on how to treatthe disease and will significantly improve individual health outcome.

Despite considerable research into methods for therapy and diseasetreatment, prostate cancer remains difficult to treat. Current methods,commonly based on surgery and/or radiation therapy, are ineffective in asignificant number of cases. Prostate surgery, for example, holds thepotential for damaging nerve tissue and compromising an individual'schances of recovering sexual function. There is a need for an imagingtechnology that can help to minimize the risks involved in surgery bydetermining the location of both the cancer and the individual's normalstructures.

Furthermore, a new technology that is able to localize cancerousprostate cells that remain following radical prostatectomy would assistphysicians in removing all of the cancerous cells from an individual'sbody with focused treatment such as radiation therapy. A labeledtechnology that selectively binds prostate cancer cells will allowclinicians to localize any remaining cancer cells following surgery. Anadditional new technology would provide direct delivery of therapeuticagents, perhaps preventing the need for surgery.

Thus, there is a need for an improved method to detect and/or diagnoselymph node metastases in individuals at risk for extraprostatic diseasefollowing the detection of elevated PSA and/or abnormal DRE and apositive biopsy.

A substantial amount of work has been put into identifying enzyme orantigen markers, which could be used as sites for detection and/ordiagnosis for various types of cancers. These markers could also be usedto target cancer cells for treatment with therapeutic and/or cancer cellkilling agents. The ideal cancer marker would exhibit, among othercharacteristics, tissue or cell-type specificity.

A 750 amino acid protein (FIG. 2; SEQ ID NO:22), prostate-specificmembrane antigen (PSMA), localized to the prostatic membrane has beenidentified. The complete coding sequence of the gene (FIG. 1;nucleotides 262 to 2514 of GenBank™ accession number NM_(—)004476) ispresented as SEQ ID NO:22. PSMA is an integral Type II membraneglycoprotein with a short intracellular tail and a long extracellulardomain. This antigen was identified as the result of generatingmonoclonal antibodies to a prostatic cancer cell, LNCaP (Horoszewicz etal. (1983) Cancer Res. 43:1809-1818). Israeli et al. (Israeli et al.(1993) Cancer Res. 53:227-230) describes the cloning and sequencing ofPSMA and reports that PSMA is prostate-specific and shows increasedexpression levels in metastatic sites and in hormone-refractory states.Other studies have indicated that PSMA is more strongly expressed inprostate cancer cells relative to cells from the normal prostate or froma prostate with benign hyperplasia. Current methods of targetingprostate specific membrane antigen use antibodies with bindingspecificity to PSMA. One of the first antibodies described with bindingspecificity to PSMA was 7E11 (Horoszewicz et al. (1987) Anticancer Res.7:927-936 and U.S. Pat. No. 5,162,504). Indium-labeled 7E11 localizes toboth prostate and sites of metastasis, and is more sensitive fordetecting cancer sites than either CT or MR imaging, or bone scan(Bander (1994) Sem. In Oncology 21:607-612).

One of the major disadvantages of the 7E11 antibody is that it isspecific to the portion of the PSMA molecule which is present on theinside of the cell (intracellular). Antibody molecules do not normallycross the cell membrane, unless they bind to an extracellular antigen,which subsequently becomes internalized. As such, 7E11 can not be usedto target a living prostate cell, cancerous or otherwise. The use of7E11 for detection or imaging is therefore limited to pockets of deadcells within cancers or tissues with large amounts of dead cells, whichcells render available their intracellular portion of PSMA for bindingwith this antibody.

U.S. Pat. No. 6,107,090, in the name of Neil Bander, and U.S. Pat. No.6,150,508, in the name of Gerald Murphy et al. describe numerousmonoclonal antibodies which recognize the extracellular domain of PSMA,thereby overcoming one of the major drawbacks of the 7E11 antibody.These antibodies, being able to bind to the extracellular domain of PSMAare capable of binding to living prostate cells, thereby allowing a moreeffective method of diagnosis than 7E11.

As described above, antibodies to PSMA are already in use for diagnosticpurposes. For example, PSMA is the antigen recognized by the targetingmonoclonal antibody used in ProstaScint™, U.S. Pat. Nos. 5,162,504 and5,763,202, Cytogen's imaging agent for prostate cancer.

It would be highly desirable to be provided with an improved antibodyspecific for PSMA and a method for diagnosis and/or detection ofcancerous prostate cells.

It would be highly desirable to be provided with a new prostate imagingtechnology offering accurate visualization of extraprostatic growthindicative of metastasis which would provide physicians with a tool todetermine the progression of the cancer and be extremely valuable indirecting treatment options.

It would be highly desirable to be provided with a non-invasive testthat is able to identify lymph node metastases in individuals at riskfor extraprostatic disease following the detection of elevated PSAand/or abnormal DRE and a positive biopsy.

It would be highly desirable to be provided with an imaging technologythat decreases morbidity by identifying individuals in which surgery isnot indicated.

It would be highly desirable to be provided with a new technology thatis able to localize cancerous prostate cells that remain followingradical prostatectomy which would assist physicians in removing all ofthe cancerous cells from an individual's body. In addition, it would behighly desirable to be provided with a new technology which wouldprovide direct delivery of therapeutic agents, perhaps preventing theneed for surgery.

It would be highly desirable to be provided with an improved method todetect and/or diagnose lymph node metastases in individuals at risk forextraprostatic disease following the detection of elevated PSA.

It would be highly desirable to be provided with a new prostate imagingtechnology that provides for accurate visualization of extraprostaticgrowth indicative of metastasis which would provide physicians with atool to determine the progression of the cancer and be extremelyvaluable in directing treatment options.

It would be highly desirable to be provided with novel antibodies andtheir use for detecting, imaging, staging, treating and monitoring ofprostate cancer, and/or metastasis thereof. It would also be highlydesirable to be provided with novel pharmaceutical compositions for thetreatment of prostate cancer.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide novel antibodies andtheir use for detecting, imaging, staging, treating and monitoring ofprostate cancer, and/or metastasis thereof.

Another aim of the present invention is to provide novel pharmaceuticalcompositions for the treatment of prostate cancer.

In accordance with one embodiment of the present invention there isprovided an antigen comprising an epitope of the extracellular region ofprostate specific membrane antigen (PSMA), ranging between amino acid 51to amino acid 67, amino acid 85 to amino acid 102, amino acid 104 toamino acid 118, amino acid 161 to amino acid 173, amino acid 236 toamino acid 245, amino acid 278 to amino acid 288, amino acid 345 toamino acid 354, amino acid 490 to amino acid 500, amino acid 531 toamino acid 545, amino acid 551 to amino acid 567, amino acid 608 toamino acid 619, amino acid 649 to amino acid 660, amino acid 716 toamino acid 724, or amino acid 738 to amino acid 750 which comprises anamino acid sequence selected from the group consisting of SEQ IDNOs:1-14, respectively.

Preferably the antigen of the extracellular region of PSMA of thepresent invention is from a mammal, more preferably a human.

In accordance with another embodiment of the present invention there isprovided a peptide selected from the group consisting of SEQ IDNOs:1-14.

In accordance with another embodiment of the present invention there isprovided a recombinant nucleic acid molecule comprising a sequence whichencodes a peptide of SEQ ID NOs:1-14, a variant or a fragment thereof.

A preferred recombinant nucleic acid molecule of the present inventionis DNA.

A preferred recombinant DNA molecule of the present invention isoperatively linked to an expression control sequence.

In accordance with another embodiment of the present invention there isprovided an expression vector containing the recombinant DNA molecule.

In accordance with another embodiment of the present invention there isprovided a method of expressing a recombinant DNA molecule in a cellcontaining the expression vector, comprising culturing the cell in anappropriate cell culture medium under conditions that provide forexpression of the recombinant DNA molecule by the cell.

A preferred method of expressing a recombinant DNA molecule in a cellcontaining the expression vector further comprises the step of purifyinga recombinant product of the expression of the recombinant DNA molecule.

In accordance with another embodiment of the present invention there isprovided a unicellular host transformed with a recombinant DNA moleculefor expression of a peptide of SEQ ID NOs:1-14, a variant or a fragmentthereof.

In accordance another embodiment of with the present invention there isprovided an immunogenic composition for raising antibodies specific toPSMA in a subject, which comprises a peptide selected from the groupconsisting of SEQ ID NOs:1-14 modified with an immunogenic moiety orcarrier and/or an antigen of the present invention in association with apharmaceutically acceptable carrier.

In a preferred immunogenic composition of the present invention thesubject is an animal selected from the group consisting of mammals andbirds, more preferably a human or a mouse, such as a BALB/c mouse, or arabbit.

In a preferred immunogenic composition the immunogenic moiety or carrieris selected from the group consisting of keyhole limpet hemocyanin (KLH)and bovine serum albumin (BSA).

In accordance with another embodiment of the present invention there isprovided a method of raising antibodies which bind to PSMA, whichcomprises administering an immunogenic amount of an immunogeniccomposition of the present invention, such as PSMA, an epitope of PSMA,or intact cell and/or fragment thereof exhibiting the extracellularregion of PSMA, to an animal.

In accordance with another embodiment of the present invention there isprovided a method of producing antibodies which bind to PSMA, comprisingtreating an animal with an immunogenic amount of an immunogeniccomposition of the present invention, such as PSMA, an epitope of PSMA,or intact cell and/or fragment thereof exhibiting the extracellularregion of PSMA, to produce antibodies; and isolating the antibodies fromserum of the animal.

In accordance with another embodiment of the present invention there isprovided an isolated antibody or antigen binding fragment thereof, whichbinds to an antigen of the present invention.

A preferred isolated antibody or antigen binding fragment thereof of thepresent invention is a monoclonal antibody, such as a monoclonalantibody selected from the group consisting of F34-8H12, F42-3E11,F42-17G1, F42-29B4, F42-30C1 AND F47-20F2, or a polyclonal antibody.

The binding fragment may be selected from the group consisting of a Fabfragment, a F(ab′)₂ fragment, and a Fv fragment.

In accordance with another embodiment of the present invention there isprovided a pharmaceutical composition for targeted treatment of prostatecancer, and/or metastasis with PSMA thereon, which comprises an antibodyor binding fragment thereof according to the present invention bound toa cytotoxic drug in association with a pharmaceutically acceptablecarrier, wherein the PSMA binding site of the antibody is available fortargeted binding to PSMA and the bound cytotoxic drug remainsbiologically active.

In a preferred pharmaceutical composition of the present invention thecytotoxic drug is selected from the group consisting of iodine-125,iodine-131, cyclophosphamide, taxol, IFN-alpha and IL2 and/or mixturesthereof.

In accordance with another embodiment of the present invention there isprovided a method for treating prostate cancer, and/or metastasisthereof comprising administering to an individual a pharmaceuticallyeffective amount of a pharmaceutical composition according to thepresent invention.

In a preferred method of the present invention the administering iscarried out orally, rectally, parenterally, subcutaneously,intravenously, intramuscularly, intraperitoneally, intraarterially,transdermally or by application to a mucus membrane.

In accordance with another embodiment of the present invention there isprovided a composition for detection of prostate cancer, and/ormetastasis thereof with PSMA thereon in an individual and/or in a sampleobtained therefrom, which comprises an antibody or binding fragmentthereof according to the present invention bound to a detectable labelin association with a physiologically acceptable carrier or an in vitroacceptable carrier, wherein the PSMA binding site of the antibody isavailable for binding to PSMA and the detectable label remainsdetectable.

In a preferred composition of the present invention the detectable labelis selected from the group consisting of a radioactive label, afluorescent label, a nuclear magnetic resonance active label, aluminescent label, a chromophore label, a positron emitting isotope forPET scanner, chemiluminescence label, or an enzymatic label.

In accordance with another embodiment of the present invention there isprovided a method of detecting prostate cancer cell, and/or metastasisthereof in an individual comprising administering to the individual aneffective amount of a composition according to the present invention orsubjecting a biological sample obtained from the individual to aneffective amount of the composition according to the present inventionand detecting the signal produced by the detectable label, whereindetection of the label above a certain level is indicative of thepresence of prostate cancer, and/or metastasis thereof. A preferredmethod of the embodiment of present invention further compriseslocalizing a detectable label within the individual or a sample obtainedtherefrom.

In a preferred method of the present invention a 2-dimensional and/or3-dimensional image of the individual or a sample obtained therefrom isgenerated.

In a preferred method of the present invention the method is used toindicate the location of prostate cancer, and/or metastasis thereofwithin the individual and/or sample obtained therefrom.

In accordance with another embodiment of the present invention there isprovided an assay system for detecting prostate cancer, and/ormetastasis thereof comprising a labeled antibody and/or antigen bindingfragment thereof according to the present invention.

A preferred assay of the present invention further comprises means forsemi-quantifying or quantifying an amount of antigen bound to theantibody and/or antigen binding fragment thereof, wherein an amount ofantigen bound to the antibody and/or antigen binding fragment thereofabove a predetermined level is indicative of prostate cancer, and/ormetastasis thereof.

In a preferred assay of the present invention the assay is selected fromthe group consisting of immunoassay, enzyme linked immunosorbent assay(ELISA), array-based immunoassay, array-based ELISA.

A preferred assay of the present invention further comprises means forreceiving the biological sample.

A preferred assay of the present invention further comprises amulti-well microplate including the antibody and/or antigen bindingfragment thereof in at least one well.

In a preferred assay of the present invention the antibody and/orantigen binding fragment thereof binds to a peptide selected from thegroup consisting of PSMA, an extracellular region of PSMA, a peptidecorresponding to an extracellular region of PSMA, an epitope of PSMA,and SEQ ID NOs:1-14.

In accordance with another embodiment of the present invention there isprovided a method of determining relative efficacy of a therapeuticregimen to be performed on an individual suffering from and/or beingtreated for prostate cancer, and/or metastasis thereof, the methodcomprising: (a) initially analyzing the individual or a biologicalsample obtained therefrom to determine presence of cancer-associatedantigen able to bind with the antibody and/or antigen binding fragmentthereof according to the present invention; and (b) periodicallyrepeating step (a) during treatment of the individual to determine anincrease or decrease in quantity of cancer-associated antigen present inthe sample.

In accordance with another embodiment of the present invention there isprovided a method of determining the recurrence of a prostate cancerdisease state in an individual clinically diagnosed as stabilized or ina remissive state, the method comprising analyzing the individual or abiological sample obtained therefrom to quantitate cancer-associatedantigen immunoreactive with an antibody and/or antigen binding fragmentthereof according to the present invention.

In accordance with another embodiment of the present invention there isprovided a kit for detecting prostate cancer, and/or metastasis thereofcomprising a composition according to the present invention.

In accordance with another embodiment of the present invention there isprovided a hybridoma cell line that produces a monoclonal antibody whichbinds to an antigen of the extracellular region of PSMA, ranging betweenamino acid 51 to amino acid 67, amino acid 85 to amino acid 102, aminoacid 104 to amino acid 118, amino acid 161 to amino acid 173, amino acid236 to amino acid 245, amino acid 278 to amino acid 288, amino acid 345to amino acid 354, amino acid 490 to amino acid 500, amino acid 531 toamino acid 545, amino acid 551 to amino acid 567, amino acid 608 toamino acid 619, amino acid 649 to amino acid 660, amino acid 716 toamino acid 724, or amino acid 738 to amino acid 750 which comprises anamino acid sequence selected from the group consisting of SEQ IDNOs:1-14, respectively.

For the purpose of the present invention the following terms are definedbelow.

The term “cancer” is intended to mean any cellular malignancy whoseunique trait is the loss of normal controls which results in unregulatedgrowth, lack of differentiation and ability to invade local tissues andmetastasize. Cancer can develop in any tissue of any organ. Morespecifically, cancer is intended to include, without limitation,prostate cancer, leukemia, hormone dependent cancers, breast cancer,colon cancer, lung cancer, epidermal cancer, liver cancer, esophagealcancer, stomach cancer.

The term “prostate cancer” is intended to mean an uncontrolled(malignant) growth of cells in the prostate gland, which is located atthe base of the urinary bladder and is responsible for helping controlurination as well as forming part of the semen.

The term “metastasis” is intended to mean cancer that has spread beyondthe prostate. “Metastasis” is also intended to mean the process by whichcancer spreads from one part of the body to another, the way it travelsfrom the place at which it first arose as a primary tumor to distantlocations in the body.

The term “antibody” (Ab) is intended to mean intact antibody moleculesas well as fragments, or binding regions or domains thereof (such as,for example, Fab, F(ab′)₂ and Fv fragments) which are capable of bindingan antigen. Such fragments are typically produced by proteolyticcleavage, with enzymes such as papain or pepsin. Alternatively,antigen-binding fragments can be produced through recombinant DNAtechnology or through synthetic procedures.

The term “monoclonal antibody” (mAb) is intended to mean an antibodyproduced by a single clone of cells or a cell line derived from a singlecell that has unique antigen binding characteristics or recognizes anindividual molecular target. Such antibodies are all identical and haveunique amino acid sequences.

The term “epitope” is intended to mean a molecular region on the surfaceof an antigen capable of eliciting an immune response and of combiningwith the specific antibody produced by such a response.

The term “cytotoxic compound” is intended to mean a compound, ormolecule which is capable of killing a cell.

The term “detectable label” is intended to mean a label effective atpermitting detection of a cell or portion thereof upon binding of amolecule to which the detectable label is attached to said cell orportion thereof. Alternatively, the detectable label permits detectionof a cell upon internalization of the detectable label by the cell. Adetectable label includes but is not limited to a radioactive label, afluorescent label, a nuclear magnetic resonance active label, aluminescent label, a chromophore label, a positron emitting isotope forPET scanner, chemiluminescence label, or an enzymatic label.

The term “biological sample” is intended to mean a sample obtained froman individual and includes, but is not to be limited to, any one of thefollowing: tissue, cerebrospinal fluid, plasma, serum, saliva, blood,nasal mucosa, urine, synovial fluid, microcapillary microdialysis.

The terms “treatment”, “treating” and the like are intended to meanobtaining a desired pharmacologic and/or physiologic effect, such asinhibition of cancer cell growth or induction of apoptosis of a cancercell. The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment” as used herein covers anytreatment of a disease in a mammal, particularly a human, and includes:(a) preventing a disease or condition (e.g., preventing cancer) fromoccurring in an individual who may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, (e.g.,arresting its development); or (c) relieving the disease (e.g., reducingsymptoms associated with the disease).

The terms “administering” and “administration” are intended to mean amode of delivery including, without limitation, oral, rectal,parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,intraarterial, transdermally or via a mucus membrane. The preferred onebeing orally. One skilled in the art recognizes that suitable forms oforal formulation include, but are not limited to, a tablet, a pill, acapsule, a lozenge, a powder, a sustained release tablet, a liquid, aliquid suspension, a gel, a syrup, a slurry, a suspension, and the like.For example, a daily dosage can be divided into one, two or more dosesin a suitable form to be administered at one, two or more timesthroughout a time period.

The term “therapeutically effective” is intended to mean an amount of acompound sufficient to substantially improve some symptom associatedwith a disease or a medical condition. For example, in the treatment ofcancer, a compound which decreases, prevents, delays, suppresses, orarrests any symptom of the disease would be therapeutically effective. Atherapeutically effective amount of a compound is not required to cure adisease but will provide a treatment for a disease such that the onsetof the disease is delayed, hindered, or prevented, or the diseasesymptoms are ameliorated, or the term of the disease is changed or, forexample, is less severe or recovery is accelerated in an individual.

The compounds of the present invention may be used in combination witheither conventional methods of treatment and/or therapy or may be usedseparately from conventional methods of treatment and/or therapy.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a compound of the present invention, as described herein,and another therapeutic or prophylactic agent known in the art.

It will be understood that a specific “effective amount” for anyparticular individual will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, and/or diet of the individual, time ofadministration, route of administration, rate of excretion, drugcombination and the severity of the particular disease undergoingprevention or therapy.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents (such as phosphate buffered saline buffers, water, saline),dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the complete nucleotide coding sequence for humanPSMA (nucleotides 262 to 2514 of Genebank accession number:NM_(—)004476) (SEQ ID NO:21).

FIG. 2 illustrates the complete amino acid sequence (amino acid 1 to750) of human PSMA (Genebank accession number: NP_(—)004467) (SEQ IDNO:22).

FIG. 3 illustrates reactivity of monoclonal antibodies of the presentinvention to LNCaP and various cells by ELISA.

FIG. 4 illustrates the specificity of monoclonal antibodies of thepresent invention to PSMA derived antigen peptides.

FIG. 5 illustrates Western blot detection of PSMA by monoclonalantibodies of the present invention.

FIGS. 6A to 6D illustrate immunohistochemical staining of prostatetissue (cancer or normal) in accordance with the present invention.

FIG. 7 illustrates Bio-distribution of monoclonal antibody of thepresent invention (8H12) in nude mice bearing LNCaP tumor.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided epitopes ofthe extracellular region of prostate specific membrane antigen (PSMA),ranging between amino acid 51 to amino acid 67, amino acid 85 to aminoacid 102, amino acid 104 to amino acid 118, amino acid 161 to amino acid173, amino acid 236 to amino acid 245, amino acid 278 to amino acid 288,amino acid 345 to amino acid 354, amino acid 490 to amino acid 500,amino acid 531 to amino acid 545, amino acid 551 to amino acid 567,amino acid 608 to amino acid 619, amino acid 649 to amino acid 660,amino acid 716 to amino acid 724, or amino acid 738 to amino acid 750which regions comprise an amino acid sequence selected from the groupconsisting of SEQ ID NOs:1-14, respectively.

Some epitopes were chosen based on hydrophilic character of the aminoacid sequence (SEQ ID NO:22) and the lack of glycosylation consensussites. Other sequences were selected from a rigorous analysis of PSMAsecondary structure prediction and homology modeling with the mostsimilar protein crystal structure (human transferrin receptor type 1).Regions were selected according to their apparent high solventaccessibility, flexibility, and coiled coil structure. In all cases theaim was to optimize antigenicity and sequence uniqueness such thatantibodies raised against these peptides do not likely cross-react withother proteins.

In accordance with the present invention, there is provided a peptidecorresponding to an epitope of the extracellular region of PSMA selectedfrom the group consisting of SEQ ID NOs:1-14.

Small molecules such as the peptides of the present invention areincomplete immunogens. Although they are able to react specifically withantibodies, they are unlikely to induce an immune response when they areinjected into an animal. In order to make them immunogenic in animals,small peptide sequences are covalently coupled to a carrier molecule,such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).KLH and BSA are coupled to the peptides via a cysteine amino acidresidue added to the N-terminus of the sequence of each peptide. Theresulting peptide-conjugates are used to raise polyclonal and monoclonalantibodies.

In accordance with the present invention, there is provided animmunogenic peptide or recombinant peptide or protein for raisingantibodies specific to PSMA, which comprises a peptide corresponding toan epitope of the extracellular region of PSMA modified with animmunogenic moiety or carrier.

In accordance with the present invention, there is provided a method forraising antibodies which bind to the epitopes and peptides of thepresent invention, which also have binding specificity to PSMA, such asPSMA in its native environment in LNCaP cells, or recombinant PSMA. Theantibodies, or binding portions thereof, recognize and bind to PSMA innormal, benign, hyperplastic and cancerous prostate cells. Moreover, theantibodies, or binding portions thereof recognize and bind to PSMA inliving normal, benign, hyperplastic and cancerous prostate cells. As aresult of this binding, the antibodies or binding portions thereof areconcentrated in areas with large numbers of prostate cells or portionsthereof.

Antibodies in accordance with the present invention may be produced byprocedures generally known in the art. For example, polyclonalantibodies may be produced by injecting the peptide or protein, such asPSMA or purified recombinant PSMA, alone or coupled to a suitableimmunogenic moiety or carrier into a non-human animal. After anappropriate period, the animal is bled, sera recovered and purified bytechniques known in the art. Monoclonal antibodies may be prepared, forexample, by the Kohler-Milstein technique (1975, Nature256(5517):497-497) involving fusion of an immune B-lymphocyte to myelomacells. For example, antigen as described above can be injected into miceas described above until a polyclonal antibody response is detected inthe mouse's sera. The mouse can be boosted again, its spleen removed andfusion with myeloma conducted according to a variety of methods. Theindividual surviving hybridoma cells are tested for the secretion ofantibodies which bind the extracellular region of PSMA first by theirability to bind the immunizing antigen (peptide/protein). Monoclonalantibodies are produced in large quantities by growing the hybridomaclones in vitro or in vivo.

Serum from immunized and nonimmunized (control) animals are tested forthe presence of specific antibodies in an Enzyme Linked ImmunoSorbentAssay (ELISA). For the ELISA assay each peptide is covalently coupled toa carrier molecule different than that used in the immunization phase ofthe procedure, or used uncoupled. Such a carrier molecule is, forexample, bovine serum albumin (BSA). The same N-terminal cysteine ofeach peptide used to couple to the carrier molecule used for raisingantibodies, for example KLH, is used to couple to the carrier moleculeused for the ELISA, for example BSA. There are two reasons for this.First, immunization of animals with peptide-KLH induces the productionof antibodies to both the peptide and KLH. Therefore, when screening forantibodies to the peptide it is important to eliminate the possibilityof detecting binding to the KLH carrier by using peptide linked to acarrier the immunized mice have never seen. This eliminates backgroundreactivity in the assay that may mask reactivity to the peptide ofinterest. Second, linking peptide to BSA in a similar manner as it waslinked to KLH should permit antibodies induced to the peptide byimmunization with peptide-KLH to recognize that peptide linked to theBSA carrier because its orientation is the same on each carrier surface.

The processes of the present invention encompass both whole antibodiesand the binding portions thereof. Such binding portions thereof includeFab fragments, F(ab′)₂ fragments, and Fv fragments. These antibodyfragments can be prepared by conventional procedures, such asproteolytic fragmentation as described in J. Goding, MonoclonalAntibodies: Principles and Practice, pp. 98-118, N.Y. Academic Press1983.

Preferred monoclonal antibodies in accordance with one embodiment of thepresent invention are identified in Table 1 below. These antibodies wereraised using peptide PS0215 (SEQ ID NO:8).

TABLE 1 Anti-PSMA Monoclonal Antibodies Monoclonal Antibody isotypeF34-8H12 IgG₃ K F42-3E11 IgG₁ K F42-17G1 IgG₁ K F42-29B4 IgG₁ K F42-30C1IgG₁ K F47-20F2 IgG₁ K

The antibody or binding portion thereof of the present invention can beused alone or in combination as a mixture with at least one otherantibody or binding portion thereof with binding specificity forprostate antigen not herein described.

In accordance with the present invention there is provided a monoclonalantibody or binding fragment thereof which binds to an epitope of theextracellular region of PSMA ranging between amino acid 51 to amino acid67, amino acid 85 to amino acid 102, amino acid 104 to amino acid 118,amino acid 161 to amino acid 173, amino acid 236 to amino acid 245,amino acid 278 to amino acid 288, amino acid 345 to amino acid 354,amino acid 490 to amino acid 500, amino acid 531 to amino acid 545,amino acid 551 to amino acid 567, amino acid 608 to amino acid 619,amino acid 649 to amino acid 660, amino acid 716 to amino acid 724, oramino acid 738 to amino acid 750 which regions comprise an amino acidsequence selected from the group consisting of SEQ ID NOs:1-14,respectively. Fourteen examples of peptides used to raise monoclonalantibodies developed using procedures described in detail below arepresented in Table 2.

In accordance with the present invention, there is provided a monoclonalantibody or binding fragment thereof which binds to a peptidecorresponding to an epitope of the extracellular region of PSMA selectedfrom the group consisting of SEQ ID NOs:1-14.

In accordance with the present invention, there is provided a hybridomacell line that produces a monoclonal antibody which binds to an epitopeof the extracellular region of PSMA, ranging between amino acid 51 toamino acid 67, amino acid 85 to amino acid 102, amino acid 104 to aminoacid 118, amino acid 161 to amino acid 173, amino acid 236 to amino acid245, amino acid 278 to amino acid 288, amino acid 345 to amino acid 354,amino acid 490 to amino acid 500, amino acid 531 to amino acid 545,amino acid 551 to amino add 567, amino acid 608 to amino acid 619, aminoacid 649 to amino acid 660, amino acid 716 to amino acid 724, or aminoacid 738 to amino acid 750 which regions comprise an amino acid sequenceselected from the group consisting of SEQ ID NOs:1-14, respectively.

In accordance with the present invention there, is provided a hybridomacell line that produces a monoclonal antibody which binds to a peptidecorresponding to an epitope of the extracellular region of PSMA selectedfrom the group consisting of SEQ ID NOs:1-14.

The antibody or binding fragment thereof, or mixtures thereof may beunmodified or may be linked to 1) a radioimaging agent, such as thoseemitting radiation, for detection of the prostate cancer, and/ormetastasis thereof upon binding of the antibody or binding fragmentthereof, or mixtures thereof to the antigen, or 2) a cytotoxic agent,which kills the prostate cancer, and/or metastasis thereof upon bindingof the antibody or binding fragment thereof, or mixtures thereof to theantigen. Alternatively, the cytotoxic agent is not toxic untilinternalized by the cell. Alternatively, the cytotoxic agent is toxicwhether internalized or not internalized. Treatment is effected byadministering the antibody or binding fragment thereof, or mixturesthereof to the individual under conditions which allow binding of theantibody or binding fragment thereof, or mixtures thereof to theantigen, and which binding results in the death of the cell of theprostate cancer, and/or metastasis thereof. In a preferred embodiment,administration is carried out on a living mammal. Such administrationcan be carried out orally or parenterally. In another embodiment themethod is used to prevent or delay development or progression ofprostate cancer, and/or metastasis thereof.

A cytotoxic agent of the present invention can be an agent emittingradiation, a cellular toxin (chemotherapeutic agent) and/or biologicallyactive fragment thereof, and/or mixtures thereof to allow cell killing.A cytotoxic agent such as a cellular toxin and/or biologically activefragment thereof can be a synthetic product or a product of fungal,bacterial or other microorganism, such as mycoplasma, viral etc.,animal, such as reptile, or plant origin. A cellular toxin and/orbiologically active fragment thereof can be an enzymatically activetoxin and/or fragment thereof, or can act by inhibiting or blocking animportant and/or essential cellular pathway or by competing with animportant and/or essential naturally occurring cellular component.

Cytotoxic agents emitting radiation for use in the present invention areexemplified by Yttrium-90 (Y⁹⁰), iodine-125 (I¹²⁵), iodine-131 (I¹³¹)and gamma-emitting isotopes used, for example, to destroy thyroid tissuein some individuals suffering from hyperthyroidism.

Radioimaging agents emitting radiation (detectable radio-labels) for usein the present invention are exemplified by indium-111 (In¹¹¹),technitium-99 (Tc⁹⁹), or iodine-131 (I¹³¹).

Detectable labels (non-radioactive labels) for use in the presentinvention can be a radioactive label, a fluorescent label, a nuclearmagnetic resonance active label, a luminescent label, a chromophorelabel, a positron emitting isotope for PET scanner, chemiluminescencelabel, or an enzymatic label. Fluorescent labels are exemplified byfluorescein, and rhodamine. Chemiluminescence labels are exemplified byluciferase. Enzymatic labels are exemplified by peroxidase andphosphatase.

Cellular toxins and/or biologically active fragments thereof areexemplified by chemotherapeutic agents (anti-cancer cytotoxic compounds)known in the art, for example, cyclophosphamide and taxol. Biologicalcompounds with cellular toxic effects are exemplified by Sapporin,Pseudomonas exotoxin (PE40), interferons (e.g. IFN-alpha) and certaininterleukins (e.g. IL2).

In accordance with the present invention there is provided apharmaceutical composition for targeted treatment of prostate cancer,and/or metastasis with PSMA thereon, which comprises an antibody orbinding fragment thereof, or mixtures thereof bound to a cytotoxic agentin association with a pharmaceutically acceptable carrier, wherein thePSMA binding site of the antibody is available for targeted binding ofPSMA and the cytotoxic agent remains biologically active.

In accordance with the present invention, there is provided a method ofdetecting normal, benign, hyperplastic and cancerous prostate epithelialcells, and/or metastases thereof in an individual or a biological sampleobtained therefrom, i.e., the detection may be in vivo or in vitro. Themethod: involves providing an antibody or binding fragment thereof or ormixtures thereof with binding specificity to an antigen of prostatecancer, or metastasis thereof. The antibody or binding fragment thereofor mixtures thereof is bound to a detectable label which upon binding ofthe antibody or binding fragment thereof or mixtures thereof allowsdetection of the prostate cancer, and/or metastasis thereof. Detectionis effected by administering the antibody or binding fragment thereof ormixtures thereof to the individual or by contacting a biological sampleobtained therefrom under conditions which allow binding of the antibodyor binding fragment thereof or mixtures thereof to the antigen. Prostatecancer, and/or metastasis thereof is detected by monitoring of thesignal produced by the detectable label above a predetermined baselevel, which indicates the presence of prostate cancer, and/ormetastasis thereof. In a preferred embodiment, administration is carriedout on a living mammal.

Detection of PSMA in, for example, a fluid sample obtained from anindividual is an indication that prostate cells are being lyzed. SincePSMA is not present in the extracellular fluid of healthy individuals,the detection of PSMA in a biological sample from an individual is anindication of prostate cell lysis.

In a preferred embodiment detection of the signal produced by thedetectable label is used in the generation of a 2-dimensional and/or3-dimensional image of the individual or a biological sample obtainedtherefrom. In another preferred embodiment the 2-dimensional and/or3-dimensional image is used to indicate the location of prostate cancer,and/or metastasis thereof within the individual or a biological sampleobtained therefrom.

In accordance with the present invention there is provided a compositionfor targeted detection of prostate cancer, and/or metastasis thereofwith PSMA thereon, which comprises an antibody or binding fragmentthereof or mixtures thereof bound to a detectable label in associationwith a physiologically acceptable carrier, wherein said PSMA bindingsite of said antibody is available for targeted binding of PSMA and saiddetectable label remains detectable from inside or outside a cell.

In accordance with the present invention there is provided a method ofdetecting prostate cancer, and/or metastasis thereof in an individual ora biological sample obtained therefrom comprising: administering to theindividual or a biological sample obtained therefrom an effective amountof a composition which comprises an antibody or binding fragment thereofor mixtures thereof bound to a detectable label in association with aphysiologically acceptable carrier, wherein the PSMA binding site of theantibody is available for targeted binding of PSMA and the detectablelabel remains detectable from inside or outside a cell; and detectingthe signal produced by the detectable label, wherein detection of thelabel above a certain level indicates the presence of prostate cancer,and/or metastasis thereof.

The antibody or binding fragment thereof or mixtures thereof withbinding specificity to an antigen of prostate cancer, and/or metastasesthereof of the present invention can be used and sold together withequipment, as a kit, to detect the particular label.

In accordance with the present invention there is provided an assaysystem for detecting prostate cancer, and/or metastasis thereofcomprising: means for receiving a biological sample; means for detectingpresence of antigen bound to at least one antibody or binding fragmentthereof or mixtures thereof; and means for quantifying an amount ofantigen bound to said at least one antibody or binding fragment thereofor mixtures thereof, wherein an amount of antigen bound to said at leastone antibody or binding fragment thereof or mixtures thereof above apredetermined level indicates prostate cancer, and/or metastasisthereof.

In accordance with the present invention there is provided a method ofdetermining the relative efficacy of a therapeutic regimen performed onan individual treated for prostate cancer, and/or metastasis thereof,the method comprising: initially analyzing an individual or a biologicalsample obtained therefrom to quantitate cancer-associated antigen ableto bind with at least one antibody or binding fragment thereof ormixtures thereof; and periodically repeating the previous step duringthe course of application of the therapeutic regimen to determineincrease or decrease in quantity of cancer-associated antigen present inthe sample.

In accordance with the present invention there is provided a method ofdetermining the recurrence of a prostate cancer disease state in anindividual clinically diagnosed as stabilized or in a remissive state,the method comprising: initially analyzing an individual or a biologicalsample obtained therefrom to quantitate cancer-associated antigenimmunoreactive with at least one antibody or binding fragment thereof ormixtures thereof; and periodically repeating the previous step duringthe course of application of the therapeutic regimen to determineincrease or decrease in quantity of cancer-associated antigen present inthe sample.

Regardless of whether the antibody or binding fragment thereof, ormixtures thereof of the present invention is used for treatment,detection, or imaging, it can be administered orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintranasal instillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, as an aerosol, or byapplication to mucous membranes, such as, that of the nose, throat, andbronchial tubes. It may be administered alone on with a pharmaceuticallyor physiologically acceptable carrier, excipient, or stabilizer, and canbe in solid or liquid form such as, tablet, capsule, powder, solution,suspension or emulsion.

The treatment and/or therapeutic use of the antibody of the presentinvention can be used in conjunction with other treatment and/ortherapeutic methods. Such other treatment and/or therapeutic methodsinclude surgery, radiation, cryosurgery, thermotherapy, hormonetreatment, chemotherapy, vaccines, other immunotherapies, and othertreatment and/or therapeutic methods which are regularly described.

In addition to methods of treatment and/or therapeutic use, theantibodies of the present invention, by their binding positions on thePSMA protein, can be used for epitope mapping of the architecture of thePSMA protein in epitope mapping studies. The antibodies of the presentinvention can also be used as probes for screening a library ofmolecules, agents, proteins, peptides and/or chemicals to identify amolecule, agent, protein, peptide and/or chemical. Such a library couldbe a chemical library, antibody library, phage display library, peptidelibrary or library of natural compounds. The identified molecule, agent,protein, peptide and/or chemical could be an antagonist or agonist ofPSMA.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example 1 Peptide Synthesis

Example 1 relates to the procedures whereby peptides corresponding toepitopes of the extracellular domain of PSMA are synthesized.

Table 2 shows the sequence and their location within the PSMA amino acidsequence of the 14 peptides that were synthesized by solid phase F-MOCchemistry to greater than 85% purity. Each peptide was synthesized witha single amino terminal unblocked cysteine residue. This amino acid wasused to conjugate each peptide to lysine residues in KLH and bovineserum albumin (BSA) carrier proteins using N-maleimide chemistry.

TABLE 2 Sequence of synthesized peptides SEQ Reference  ID No.Peptide Sequence^(a) Location NO 4243 NH₂-CNITPKHNMKAFLDELKA 51-67 14244 NH₂-CGTEQNFQLAKQIQSQWKE  85-102 2 PS0210 NH₂-CGLDSVELAHYDVLLS104-118 3 PS0211 NH₂-CFSAFSPQGMPEGD 161-173 4 PS0212 NH₂-CAPGVKSYPDG236-245 5 PS0213 NH₂-CAYRRGIAEAVG 278-288 6 PS0214 NH₂-CHIHSTNEVTR345-354 7 PS0215 NH₂-CGKSLYESWTKK 490-500 8 4245 NH₂-CASGRARYTKNWETNK531-545 9 4246 NH₂-CLYHSVYETYELVEKFYD 551-567 10 PS0216NH₂-CADKIYSISMKHP 608-619 11 PS0217 NH₂-C-CSERLQDFDKSNPIVLR-C 649-660 12PS0218 NH₂-CESKVDPSKA 716-724 13 PS0219 NH₂-CTVQAAAETLSEVA 738-750 14^(a)N-terminal C residues on each peptide are optionally added formanipulation and/or coupling; they are not part of the PSMA sequence.The C residues at the N-terminal and C-terminal of PS0217 also allow forthe potential for cyclization.

Example 2 Preparation of Monoclonal Antibodies

Example 2 relates to preparation of mouse monoclonal antibodies withspecificity to the peptides of Example 1.

Several strategies were used to immunize BALB/c mice for production ofPSMA-specific antibodies.

One strategy consisted of priming and boosting at 2 to 3 week intervalswith peptide conjugated to KLH by one of 2 methods that link the aminoterminal cysteine of the peptide immunogen to lysine residues on KLH.Peptides were conjugated to KLH using either sulfo-GMBS or SMCCconjugation systems. This strategy was designed to induce and amplifypeptide specific antibodies.

A second strategy employed 2 immunizations at 2 to 3 week intervals withLNCaP membrane followed by 3 immunizations with purified PSMA or peptideconjugated KLH. Priming with LNCaP membrane should induce the productionof an antibody response directed to membrane antigens including PSMApresented in a native conformation within a cellular membrane. Boostingwith purified PSMA antigen should further activate and expand the Blymphocyte clones secreting antibody that recognizes epitopes present onwhole native PSMA whereas boosting with peptide conjugated KLH shouldfurther activate and expand the B lymphocyte clones recognizing theepitopes corresponding to the peptide used in the boost immunizations.

All immunizations were intraperitoneal injections of 100 μl volumescontaining 25 to 50 μg of peptide antigen or 50 μl of LNCap membranepreparation. The antigen for the first immunization was emulsified incomplete Freund's adjuvant (CFA). Antigen used for subsequentimmunizations was emulsified in incomplete Freund's adjuvant (IFA). Thefinal boost before fusing donor spleen with the NSO myeloma parentalcell line was done 3 to 5 days before fusion. For this immunizationantigen was diluted in phosphate buffered saline (PBS).

The fusion was performed according to the technique known in the art(Kohler G. and Milstein C. (1975) Nature 256 (5517):495-97).

Supernatants of the resulting wells exhibiting growth were screened byEnzyme Linked Immunosorbent Assay (ELISA) for the presence of antibodiesbinding to peptide (conjugated or not to BSA) and either LNCaP cellmembranes or recombinant PSMA. Negative controls for the screening stepwere BSA alone (control for peptide or PSMA binding) or PC-3 cellmembrane (control for LNCaP binding). Wells containing antibodies withdesirable binding characteristics were subjected to at least 2 cycles ofcloning by limiting dilution. Hybridomas secreting either one of the 6monoclonal antibodies against peptide PS0215 (SEQ ID NO:8) weregenerated according to this screening strategy. The isotype of theimmunoglobulin secreted into cultured supernatants by cloned antibodysecreting hybridomas was determined using Isostrips (Roche DiagnosticsCorp., Indianapolis Ind.).

Example 3 Preparation of Cell Membrane and Purified PSMA Cell MembranePreparation

Example 3 relates to the purification of recombinant PSMA and cellmembrane for immunization and characterization of mAb.

LNCaP cells (ATCC No. ERL-1740), PC3 (ATCC No. CRL 1435 KS62 (ATCC No.CCL 243), NMB7 (Gift from Dr. U. Saragovi) were grown at 37° C. inRPMI-1640 supplemented with 10 mM HEPES, 10% FCS, 30 μg/ml kanamycin,200 μg/ml streptomycin, and 20 μg/ml neomycin, and 2 mM L-glutamine,under a humidified atmosphere of 5% CO₂. When confluent, cells werewashed with PBS and detached using 1 mM EDTA in PBS. Cells were spundown and the pellet frozen. Packed cells were resuspended in 10 volumesof ice cold hypotonic buffer (5 mM Tris pH 7.6; 2 mM EDTA) containingprotease inhibitors (20 μg/ml of TLCK (Nα-p-tosyl-1-lysine chloromethylketone) 20 μg/ml TPCK (N-tosyl-1-phenylalanine chloromethyl ketone) and20 μg/ml PMSF (phenylmethyl sulfonyl fluoride). Cells were sonicatedusing a probe sonicator at medium setting with three 30-second bursts onice. Sonicated cells were centrifuged at 1500×g for 10 min at 4° C.Supernatant was collected and centrifuged at 12,000×g for 60 min at 4°C. The membrane pellet was resuspended in 10 volumes of the followingbuffer (250 mM sucrose, 50 mM Tris-HCl pH7.4, 5 mM EDTA, 100 mM NaCl)and frozen until use.

Cloning of PSMA from LNCaP Cells

Total RNA from LNCaP was isolated using the Trizol method according tomanufacturer's directions (GIBCO Life Technologies Inc.) and treatedwith DNase I (RNase free). LNCaP RNA was reverse transcribed byThermoscript reverse transcriptase and oligo dT primers (GIBCO LifeTechnologies Inc.). DNA corresponding to the gene encoding PSMA was thenamplified by PCR using the oligonucleotides (5′3′)ATGTGGAATCTCCTTCACGAAACC (SEQ ID NO:15) and TTAGGCTACTTCACTCAAAGTCTC(SEQ ID NO:16). The resulting PCR product was cloned into plasmidpCRT7-NT. Clones were sequenced to verify the identity of the insert DNAas originating from PSMA.

Baculovirus Expression of PSMA

PSMA was PCR-amplified from a sequence-confirmed recombinant plasmid ofpCRT7-NT using primers GGGGATCCATGTGGAATCTCCTTCACG (SEQ ID NO:17) andGGGCTCGAGGGCTACTTCACTCAAAGTCT (SEQ ID NO:18) (full length PSMA, fIPSMA)or the oligonucleotides GGGGATCCGAAATCCTCCAATGAAGCTACTAAC (SEQ ID NO:19)and GGGCTCGAGTTAGGCTACTTCACTCAAAGTCTC (SEQ ID NO: 20) (soluble PSMA,sPSMA). The PCR fragment was digested overnight with the restrictionenzymes BamHI and XhoI and cloned into Novagen transfer vector pBAC-1(fIPSMA) or pBAC-3 (sPSMA). The recombinant virus encoded either a fulllength PSMA containing a C-terminal poly-histidine tag or a truncatedPSMA containing a poly-histidine tag at the N-terminus. Sf9 cells wereco-transfected with the transfer vector DNA and the linearized viral DNAaccording to the manufacturer's directions. The viruses were plaquepurified prior amplification to obtain a high titer viral stock.

Sf9 cells were propagated in TNM-FH medium supplemented with 10% fetalbovine serum, 0.1% Pluronic F-68 (InVitrogen), and the antibioticskanamycin (30 ug/ml), neomycin (20 ug/ml) and streptomycin (200 ug/ml).Infection of Sf9 cells with recombinant baculovirus was done at amultiplicity of infection of about 10. After 3 days post-infection.flPSMA was solubilized from a cell lysate (PBS containing 1% TritonX-100) and secreted sPSMA was recovered directly from the medium. Bothproteins were purified by affinity chromatography using a Ni-NTA resin,according to the manufacturer's instruction (Qiagen). The eluate wasdialysed extensively against PBS before use as an immunogen or forhybridoma screening.

Example 4 Characterization of Monoclonal Antibodies MonoclonalAntibodies Reactivity to PSMA by ELISA

Example 4 relates to the characterization of the mAbs by ELISA, westernblot IHC, and in vivo biodistribution.

mAb reactivity to PSMA was assayed by ELISA. The LNCaP cell line wasused as a source of natural PSMA and various PSMA non-expressing cellline as negative control. 5 ug of cell membrane preparation in 100 ulPBS were adsorbed onto 96 well plates (Immulon 2HB, Thermo Labs System)overnight at 4° C., or 2 hours at room temperature. The plates werewashed with TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20)then incubated with TBST containing 3% casein for 1 hours to blocknon-specific sites. The wells were loaded with 100 ul of the hybridomacell supernatants or a dilution in TBST, and incubated for 1 hour atroom temperature under gentle agitation. In some cases, the mAb waspre-mixed with dilutions of the antigenic peptide or an irrelevantpeptide and then the solution applied to coated cell lysate. The plateswere washed with TBST then incubated for 1 hour with a horse-radishperoxidase conjugated goat anti-mouse IgG (Jackson #115-035-164)secondary antibody at a dilution 1/1000 in TBST. After extensivewashing, the plates were incubated with 100 ul of the peroxidasesubstrate TMB (BioFX). The reaction was stopped with an equivalentvolume of 0.5N sulfuric acid and the reactivity evaluated by reading atOD 450 nm.

FIG. 3 shows a representative reactivity of the six monoclonalantibodies for the LNCaP cells (-□-) compared to the PSMA negative humancancer cell lines PC-3 (prostate, -Δ-), K562 (myeloid leukemia, -x-) andNMB-7-(neuroblastoma, -Δ-). The graph illustrates that only a very weaksignal was detected from the negative control cell lines as compared tothe strongly reactive LNCaP cells. Indeed, the average reactivity (±SEM)of the antibodies to LNCaP over PC-3 background was found to be 9.0±3.6for the 8H12 (n=8), 25.7±6.3 for the 3E11 (n=7), 26.1±6.32 for the 29B4(n=8), 10.9±3.0 for the 30C1 (n=5), 16.9±4.4 for the 17G1 (n=5), and58.9±15.6 for the 20F2 (n=4). These results suggest that the reactivityof the mAbs is specific for a protein expressed by the LNCaP cells only.

In order to confirm the specificity of the mAbs, the reactivity of themAbs to LNCaP cells were challenged by the original antigen from whichthey were generated (PS0215) (SEQ ID NO:8). FIG. 4 shows that nanomolarconcentrations of the antigenic peptide PS0215 (-□-) can completelyinhibits the binding of the antibodies to LNCaP cells. In contrast, nochange in the reactivity of the antibodies were observed when challengedwith up to micromolar concentration of another peptide derived from thePSMA amino acid sequence (PS0210, -∘-). The results suggests that theantibodies recognize a unique linear amino acid sequence of PSMA(PS0215) i.e. corresponding to PS0215 or SEQ ID NO:8.

Western Blot Detection of PSMA

Western Blot analysis were performed on LNCaP and PC-3 cell membrane inorder to confirm that the mAbs detect the PSMA protein. Proteins from2.5 ug of a cell membrane preparation were separated bySDS-polyacrylamide gel electrophoreisis on a 7.5% gel. The proteins werethen transferred to a PVDF membrane and the membrane was blocked with 3%casein in TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) for1 hour at room temperature. After washing, the membrane was incubatedwith the hybridoma supernatant diluted 1/1000 in TBST, and incubated 1hour under gentle agitation. After extensive washing with TBST, themembrane was incubated with a 115000 dilution of horse-radish peroxidaseconjugated goat anti-mouse IgG (Jackson #115-035-164) secondary antibodyfor 1 hour. After washing, the membrane was developed with achemiluminescent substrate according to the manufacturer'srecommendations (Pierce #34080).

FIG. 5 shows that all mAbs detected a single band of a molecular weightof about 100 KDa in LNCaP cell membrane (lane 1) and not in the PC-3cell membrane (lane 2). The fact that the antibodies detected a bandfrom a reducing and denaturing gel also confirm that they recognise alinear amino acid sequence of PSMA as opposed to a conformationalepitope.

Immunohistochemical Staining of Prostate Cancer Tissue

Immunohistochemical staining was performed on paraffin embedded sectionfrom prostate cancer. After deparafinization and rehydration throughgraded alcohol, endogenous peroxidase was inactivated by treatingsections with 3% H₂O₂ for 20 min at RT. Non specific binding was blockedwith 5% normal goat serum (NGS) in 0.01 M phosphate buffered saline pH7.4; 0.05% Triton (PBS-T) for 30 min at RT before adding primaryantibodies diluted in PBS-T; 2% NGS overnight at RT. 8H12 was used as atissue culture supernatant diluted 1:5. Mouse IgG with an irrelevantspecificity was used as a negative control at a concentration of 2μg/ml. After washing, binding of primary antibody to tissue sections wasdetected by sequential addition followed by washing of goat anti-mouseIg heavy+light chain polyclonal antibody (ICN) at 1:100, a complex ofhorse radish peroxidase (HRP, 5 μg/ml) and a mouse monoclonal antibodyengineered to have dual specificity for goat antibody and HRP (1/30),and DAB (0.06%); 0.01% H₂O₂ all diluted in PBS-T; 2% NGS. Sections werewashed in tap water, counterstained with hematoxylin and rinsed in tapwater. Sections were then dehydrated and mounted in Permount™ (Sigma). Apathologist evaluated all immunohistochemical sections in a blindedfashion.

FIGS. 6A to D show paraffin embedded sections of prostate tissue frompatients diagnosed with prostate cancer, stained immunohistochemicallywith the mAb 8H12. Shown are results for non antigen retrieved material.While 8H12 bound PSMA focally in prostate epithelial cells of bothbenign and malignant prostate tissue, normal structures in theprostatric stroma, nerve tissue, smooth muscles of blood vessel wallsand collagen, were negatively stained for PSMA (FIG. 6A). As well,inflammatory cells (not shown) and endothelial cells stain negatively.

Staining of the benign prostatic glands, composed of prostatic acinarcells and underlying basal cells, show that the basal cells are PSMAnegative, whereas the acinar cells are PSMA positive, mainly at theluminal aspect of the plasma membrane (FIGS. 6B, C and D). 8H12 showsmoderate staining of PSMA in well differentiated prostate cancer, i.e.Gleason 3+3=6. Weaker cytoplasmic staining is also seen.

In Vivo Biodistribution of Labeled Anti-PSMA mAbs

Purification of mAb: Cells were grown in Iscove's medium, 20% FCS, IL-6(1 mg/ml), and antibiotics using T175 flasks. After reaching confluence,cells were removed by centrifugation. The medium was precipitated withsaturated ammonium sulfate (final concentration=45%) overnight at 4° C.The solution was centrifuged and the supernatant discarded. Theprecipitate was resuspended in PBS pH 7.4 and further dialyzed againstPBS at 4° C. A 5 ml protein G column (Amersham) was equilibrated with 20mM NaH₂PO₄ pH 7.0 and the Ab solution was then passed through using asyringe barrel. The column was washed with 20 mM NaH₂PO₄ pH 7.0 andfinally elution was done using Pierce's ImmunoPure Gentle Ag/Ab Buffer.Fractions containing the Ab were combined and buffer exchanged into PBSusing Amicon Centriplus filtration devices.

Labelling of mAbs: 100 ug mAb were labelled by the method of chloramineT (Bioconjugate Techniques (1996) Elsevier Science (USA)) by mixingabout 10 mCi NaI¹²⁵ and five fold antibody molar equivalent ofchloramine T in a total volume of 135 ul. After 30 seconds, the reactionwas quenched with 100 ul sodium meta-bisulfite at a concentration of 2.6mg/ml. Free I¹²⁵ was removed by gel filtration of the antibody solutionin a sodium phosphate buffer containing 0.1% BSA. 85% to 92% of theradioactive iodine was associated with the antibody, as assessed byHPTLC.

In Vivo Biodistribution of Labelled Anti-PSMA Mabs

In vivo targeting potential of the I¹²⁵-8H12 and I¹²⁵-29B4 was assessedin nude mice bearing LNCaP and/or PC3 tumors. Nude mice were injectedsubcutaneously in the flank with 0.5×10⁶ trypsinized LNCaP cells and/orin the other flank with PC-3 cells in a volume of 200 ul PBS containing50% Matrigel (Becton Dickinson). 1 month after the cell injection, themice were administered, by tail vein injection, 2 or 20 ug of thementioned labelled mAb at a specific activity of ˜2 uCi/ug. After 24 or48 hours post-injection, the mice were sacrificed and the tumors andmajor organs were recovered and cleaned from blood. A blood sample wasalso obtained at the time of sacrifice. The blood and tissue sampleswere weighted and counted for radioactivity incorporation in a gammacounter.

The relative activity of the tissue (cpm) was expressed per mg oftissue. For mice bearing both LNCaP and PC-3 tumors, the ratio of therelative activity of LNCaP/PC-3 tumor was calculated. For comparison ofmAb uptake between mice, relative tissue activity was first normalizedto blood to account for difference in the efficiency of injection, andthen the ratio of the relative activity of LNCaP tumor over non tumortissue was calculated.

FIG. 7 shows the LNCaP retention potential of the labeled Ab over normaltissue 48 hrs after an injection. The LNCaP tumor retained the labelled8H12 antibody between 2.7 and 6.5 times better than the various tissuestested. The tissue retention was comparable at 24 h post-injection,indicating a complete bio-distribution of the mAb in a minimum of 24 h.These results indicate a significant concentration of 8H12 in LNCaPtumor compared to major organs.

The selectivity of the 8H12 and 29B4 for LNCaP tumor compared to PC-3tumor was also measured in mice bearing both type of cells. Table 3shows that Zug of the labelled 81-112 resulted in the concentration ofthe mAb 4.3 times higher than in the PC-3 tumor.

TABLE 3 In vivo tumor selectivity of anti-PSMA mAb LNCaP/PC-3 tumorratio, 48 hrs post injection mAb Ratio  2 μg 8H12 4.3 20 μg 29B4 2.7 20ug of the mAb 29B4, also revealed a significant concentration (2.7times) in LNCaP tumor compared to PC-3.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. An antigen comprising an epitope of the extracellular region ofprostate specific membrane antigen (PSMA), ranging between amino acid 51to amino acid 67, amino acid 85 to amino acid 102, amino acid 104 toamino acid 118, amino acid 161 to amino acid 173, amino acid 236 toamino acid 245, amino acid 278 to amino acid 288, amino acid 345 toamino acid 354, amino acid 490 to amino acid 500, amino acid 531 toamino acid 545, amino acid 551 to amino acid 567, amino acid 608 toamino acid 619, amino acid 649 to amino acid 660, amino acid 716 toamino acid 724, or amino acid 738 to amino acid 750 which comprises anamino acid sequence selected from the group consisting of SEQ IDNOs:1-14, respectively.
 2. The antigen of claim 1, wherein said PSMA isfrom a mammal.
 3. The antigen of claim 1, wherein said PSMA is from ahuman.
 4. A peptide selected from the group consisting of SEQ IDNOs:1-14.
 5. A recombinant nucleic acid molecule comprising a sequencewhich encodes a peptide of claim 4, a variant or a fragment thereof.6-60. (canceled)
 61. A method of producing antibodies or antigen bindingfragments thereof for binding to a prostate specific membrane antigenPSMA comprising SEQ ID NO:22, said method comprising: a) treating ananimal with an immunogenic amount of an immunogenic compositioncomprising a peptide selected from the group consisting of SEQ ID NO.:1,SEQ ID NO.:2, SEQ ID NO.:3, SEQ ID NO.:4, SEQ ID NO.:5, SEQ ID NO.:6,SEQ ID NO.:7, SEQ ID NO.:9, SEQ ID NO.:10, SEQ ID NO.:11, SEQ ID NO.:12,SEQ ID NO.:13 and SEQ ID NO.:14 coupled to an immunogenic moiety orcarrier to produce antibodies; b) isolating said antibodies of step a)from serum of said animal; and optionally: c) producing antigen bindingfragments of said isolated antibodies.
 62. An isolated antibody orantigen binding fragment thereof, which specifically binds to a peptideselected from the group consisting of SEQ ID NO.:1, SEQ ID NO.:2, SEQ IDNO.:3, SEQ ID NO.:4, SEQ ID NO.:5, SEQ ID NO.:6, SEQ ID NO.:7, SEQ IDNO.:9, SEQ ID NO.:10, SEQ ID NO.:11, SEQ ID NO.:12, SEQ ID NO.:13 andSEQ ID NO.:14 and which specifically binds to an epitope of a prostatespecific membrane antigen comprising SEQ ID NO:22.
 63. The isolatedantibody or antigen binding fragment thereof according to claim 62,wherein the antigen binding fragment is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, and a Fv fragment. 64.A pharmaceutical composition for targeted treatment of prostate cancerexpressing a PSMA polypeptide comprising SEQ ID NO.:22 and/or metastasesthereof, the pharmaceutical composition comprising the antibody orantigen binding fragment thereof according to claim 62, linked to acytotoxic drug and a pharmaceutically acceptable carrier, wherein saidantibody is available for targeted binding to said PSMA polypeptide andsaid linked cytotoxic drug remains biologically active.
 65. Thepharmaceutical composition according to claim 64, wherein the at leastone cytotoxic drug is selected from the group consisting of Iodine-125,Iodine-131, cyclophosphamide, Yttrium-90, paclitaxel, IFN-alpha, andIL-2.
 66. The pharmaceutical composition according to claim 65, whereinsaid antibody is a monoclonal antibody.
 67. A composition for detectionof prostate cancer expressing a PMSA polypeptide comprising SEQ IDNO.:22 and/or metastases thereof in an individual and/or in a sampleobtained therefrom, which comprises the antibody or antigen bindingfragment thereof according to claim 62 adapted to be linked to adetectable label in association with a physiologically acceptablecarrier or an in vitro acceptable carrier, wherein said antibody isavailable for binding and said detectable label when linked to saidantibody remains detectable.
 68. The composition according to claim 67,wherein said detectable label is selected from the group consisting of aradioactive label, a fluorescent label, a nuclear magnetic resonanceactive label, a luminescent label, a chromophore label, a positronemitting isotope for PET scanner, a chemiluminescence label and anenzymatic label.
 69. An isolated antibody or antigen binding fragmentthereof for binding to an epitope of a PSMA polypeptide comprising SEQID NO.:22, said antibody or antigen binding fragment thereof being anantibody produced by the process of immunizing an animal with animmunogenic amount of an immunogenic composition comprising a peptideselected from the group consisting of SEQ ID NO.:1, SEQ ID NO.:2, SEQ IDNO.:3, SEQ ID NO.:4, SEQ ID NO.:5, SEQ ID NO.:6, SEQ ID NO.:7, SEQ IDNO.:9, SEQ ID NO.:10, SEQ ID NO.:11, SEQ ID NO.:12, SEQ ID NO.:13 andSEQ ID NO.:14 coupled to an immunogenic moiety or carrier and optionallyisolating said antibody and producing said antigen binding fragmentsthereof from the isolated antibody.
 70. The isolated antibody or antigenbinding fragment thereof, according to claim 69, wherein the antibody isa monoclonal antibody or a polyclonal antibody.
 71. The isolatedantibody or antigen binding fragment thereof, according to claim 69,wherein the antigen binding fragment is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment and a Fv fragment. 72.A pharmaceutical composition comprising the antibody of claim 69, and apharmaceutically acceptable carrier.
 73. The pharmaceutical compositionof claim 72, wherein the antibody is conjugated to a cytotoxic drug. 74.The pharmaceutical composition of claim 73, wherein the cytotoxic drugis selected from the group consisting of Iodine-125, Iodine-131,cyclophosphamide, Yttrium-90, paclitaxel, IFN-alpha and IL-2.
 75. Thepharmaceutical composition of claim 72, wherein the antibody isconjugated to a detectable label.
 76. The pharmaceutical composition ofclaim 75, wherein the detectable label is selected from the groupconsisting of a radioactive label, a fluorescent label, a nuclearmagnetic resonance active label, a luminescent label, a chromophorelabel, a positron emitting isotope for PET scanner, a chemiluminescencelabel and an enzymatic label.